JP6441599B2 - Production method of pravastatin - Google Patents
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Description
本発明は、微生物を利用したプラバスタチンの新たな製造法に関する。 The present invention relates to a new method for producing pravastatin using microorganisms.
プラバスタチンナトリウムは、コレステロール生合成酵素の一つであるＨＭＧ−ＣｏＡリダクターゼ阻害作用を有し、高コレステロール血症治療薬として世界中で広く使用されている。プラバスタチンは、種々の微生物によりコンパクチンからプラバスタチンへ変換されて製造され、当該微生物としてはストレプトマイセス属、アスペルギルス属、モナスカス属、ペニシリウム属、アクチノマデュラ属が知られている（特許文献１〜５）。また、マイクロモノスポラ属に属する菌により、コンパクチンからプラバスタチンが得られることも報告されている（特許文献６、７）。 Pravastatin sodium has an action of inhibiting HMG-CoA reductase, which is one of cholesterol biosynthesis enzymes, and is widely used all over the world as a therapeutic agent for hypercholesterolemia. Pravastatin is produced by converting compactin to pravastatin by various microorganisms, and as the microorganism, Streptomyces, Aspergillus, Monascus, Penicillium, and Actinomadura are known (Patent Documents 1 to 5). . It has also been reported that pravastatin can be obtained from compactin by bacteria belonging to the genus Micromonospora (Patent Documents 6 and 7).
しかしながら、従来の微生物によるコンパクチンからプラバスタチンへの変換効率は十分でなく、さらに新たな微生物によるプラバスタチンの製造法が望まれている。
従って、本発明の課題は、新たな微生物を利用したプラバスタチンの製造法を提供することにある。
However, the conversion efficiency from compactin to pravastatin by conventional microorganisms is not sufficient, and a new method for producing pravastatin by microorganisms is desired.
Accordingly, an object of the present invention is to provide a method for producing pravastatin using a new microorganism.
そこで本発明者は、プラバスタチン生産能を有する微生物を求めて種々検討したところ、全く意外にも、アクチノプラネス属に属する微生物の中にコンパクチンをプラバスタチンに変換する能力が優れた微生物が複数存在することを見出し、本発明を完成した。 Therefore, the present inventor conducted various studies in search of microorganisms capable of producing pravastatin, and surprisingly, there are a plurality of microorganisms excellent in the ability to convert compactin to pravastatin among microorganisms belonging to the genus Actinoplanes. The present invention has been completed.
すなわち、本発明は、次の〔１〕〜〔３〕の発明を提供するものである。
〔１〕アクチノプラネス属に属し、コンパクチンをプラバスタチンに変換する能力を有する微生物を含有する培地に、コンパクチン又はその塩を添加して培養することを特徴とするプラバスタチン又はその塩の製造法。
〔２〕アクチノプラネス属に属し、コンパクチンをプラバスタチンに変換する能力を有する微生物が、ＲＤ０６１８０４（ＮＩＴＥ Ｐ-０１８７４）又はこの菌株と１６ＳｒＲＮＡの塩基配列の同一性が９３％以上のアクチノプラネス属細菌である〔１〕記載のプラバスタチン又はその塩の製造法。
〔３〕アクチノプラネス属に属し、コンパクチンをプラバスタチンに変換する能力を有する微生物が、ＲＤ０６１８０４（ＮＩＴＥ Ｐ-０１８７４）、アクチノプラネス コイキイ、アクチノプラネス ロバタス、アクチノプラネス イタリカス、アクチノプラネス レギュラリス、アクチノプラネス フィリピネンシス、アクチノプラネス ブラジリエンシス及びアクチノプラネス テイコマイセチカスから選ばれる微生物である〔１〕又は〔２〕記載のプラバスタチン又はその塩の製造法。
That is, the present invention provides the following inventions [1] to [3].
[1] A method for producing pravastatin or a salt thereof, which comprises culturing by adding compactin or a salt thereof to a medium containing a microorganism belonging to the genus Actinoplanes and having the ability to convert compactin to pravastatin.
[2] A microorganism belonging to the genus Actinoplanes and having the ability to convert compactin to pravastatin is RD061804 (NITE P-01874) or a bacterium belonging to the genus Actinoplanes having a 16S rRNA base sequence identity of 93% or more [1] A process for producing pravastatin or a salt thereof according to [1].
[3] Microbes belonging to the genus Actinoplanes and having the ability to convert compactin to pravastatin are RD061804 (NITE P-01874), Actinoplanes Koikii, Actinoplanes Lobatas, Actinoplanes Itaricas, Actinoplanes regularis, Actinoplanes filipinensis The method for producing pravastatin or a salt thereof according to [1] or [2], which is a microorganism selected from Actinoplanes bragiliensis and Actinoplanes Teikomyceticus.
本発明方法によれば、コンパクチンから良好な収率で高コレステロール血症治療薬として有用なプラバスタチン又はその塩を製造できる。 According to the method of the present invention, pravastatin or a salt thereof useful as a therapeutic agent for hypercholesterolemia can be produced from compactin in good yield.
本発明のプラバスタチン又はその塩の製造法は、アクチノプラネス属に属し、コンパクチンをプラバスタチンに変換する能力を有する微生物を含有する培地に、コンパクチン又はその塩を添加して培養することを特徴とする。 The method for producing pravastatin or a salt thereof of the present invention is characterized by adding compactin or a salt thereof to a medium containing a microorganism belonging to the genus Actinoplanes and having the ability to convert compactin to pravastatin.
アクチノプラネス属に属し、コンパクチンをプラバスタチンに変換する能力を有する微生物としては、ＲＤ０６１８０４（ＮＩＴＥ Ｐ-０１８７４）、及びこの菌株と１６ＳｒＲＮＡの塩基配列の同一性が９３％以上のアクチノプラネス属細菌が挙げられ、ＲＤ０６１８０４（ＮＩＴＥ Ｐ-０１８７４）、及びこの菌株と１６ＳｒＲＮＡの塩基配列の同一性が９５％以上のアクチノプラネス属細菌が好ましく、ＲＤ０６１８０４（ＮＩＴＥ Ｐ-０１８７４）、及びこの菌株と１６ＳｒＲＮＡの塩基配列の同一性が９８％以上のアクチノプラネス属細菌がより好ましい。 Examples of microorganisms belonging to the genus Actinoplanes and having the ability to convert compactin to pravastatin include RD061804 (NITE P-01874), and bacteria belonging to the genus Actinoplanes having a nucleotide sequence identity of 16% or more with this strain. , RD061804 (NITE P-01874), and Actinoplanes genus bacteria having an identity of 95% or more of the base sequence of 16SrRNA with this strain, RD061804 (NITE P-01874), and the identity of the base sequence of 16SrRNA with this strain are preferred. Actinoplanes bacteria having a sex of 98% or more are more preferable.
前記菌株と１６ＳｒＲＮＡの塩基配列の同一性が９３％以上のアクチノプラネス属細菌としては、アクチノプラネス コイキイ（Ａｃｔｉｎｏｐｌａｎｅｓ ｃｏｉｃｈｉｉ）、アクチノプラネス ロバタス（Ａｃｔｉｏｎｐｌａｎｅｓ ｌｏｂａｔｕｓ）、アクチノプラネス イタリカス（Ａｃｔｉｏｎｐｌａｎｅｓ ｉｔａｌｉｃｕｓ）、アクチノプラネス レギュラリス（Ａｃｔｉｏｎｐｌａｎｅｓ ｒｅｇｕｌａｒｉｓ）、アクチノプラネス フィリピネンシス（Ａｃｔｉｏｎｐｌａｎｅｓ ｐｈｉｌｉｐｐｉｎｅｎｓｉｓ）、アクチノプラネス ブラジリエンシス（Ａｃｔｉｏｎｐｌａｎｅｓ ｂｒａｓｉｌｉｅｎｓｉｓ）、アクチノプラネス テイコマイセチカス（Ａｃｔｉｏｎｐｌａｎｅｓ ｔｅｉｃｈｏｍｙｃｅｔｉｃｕｓ）等が挙げられる。 Actinoplanes coikii, Actinoplanes lobatus, Actinoplanes italica, Actinoplanes actiplanes, Actinoplanes actiplanes, Actiplanas Actionplanes legalis), Actinoplanes philippinensis (Actionplanenes philippinensis), Actinoplanes brasiliensis (Actinoplanes brasiliensis), Actinoplanes teicomyceticus (Actinoplanet teikomyceticus) It is done.
ＲＤ０６１８０４（ＮＩＴＥ Ｐ-０１８７４）の菌学的性質は、次のとおりである。
（１）比較的大きく、シワ状の硬いコロニーを形成する。色はオレンジ〜やや茶色である。胞子は形成しない。
（２）ＩＳＰ−２寒天培地で良好に生育する。ＩＳＰ−１、ＩＳＰ−３、ＩＳＰ−４、ＩＳＰ−５、ＩＳＰ−６、Ｖ８ジュース寒天培地及びＢｅｎｎｅｔｔ’ｓ−マルトース培地で中程度に増殖する。ＩＳＰ−７、ＩＳＰ−９、ＨＶ及びＹＳ−Ｂ寒天培地で僅かに増殖する。
（３）生育炭素源として、グルコースよりもマルトースの方をよく好み、増殖する傾向がある。
The mycological properties of RD061804 (NITE P-01874) are as follows.
(1) A relatively large and wrinkled hard colony is formed. The color is orange to slightly brown. Spores do not form.
(2) It grows well on ISP-2 agar medium. Grow moderately on ISP-1, ISP-3, ISP-4, ISP-5, ISP-6, V8 juice agar and Bennett's-maltose media. Grows slightly on ISP-7, ISP-9, HV and YS-B agar.
(3) As a growth carbon source, maltose is preferred over glucose and tends to proliferate.
本発明の製造法には、アクチノプラネス属に属し、コンパクチンをプラバスタチンに変換する能力を有し、上記（１）〜（３）の菌学的性質を有する菌を好ましく用いることができる。 In the production method of the present invention, a bacterium belonging to the genus Actinoplanes, having the ability to convert compactin to pravastatin, and having the above-mentioned mycological properties (1) to (3) can be preferably used.
本発明に使用する株は、前記の株はもとより、その人工変異株あるいは自然変異株も、コンパクチンをプラバスタチンに変換する能力を有するものであれば使用可能である。人工変異株は、例えば紫外線照射、コバルト６０照射、または化学変異誘発剤等により容易に得ることが出来る。 As the strain used in the present invention, not only the aforementioned strains but also artificial mutants or natural mutants thereof can be used as long as they have the ability to convert compactin into pravastatin. The artificial mutant can be easily obtained by, for example, ultraviolet irradiation, cobalt 60 irradiation, or a chemical mutagen.
本発明方法に用いられる培地としては、アクチノプラネス属の菌が生育する培地であればよく、例えばグルコース、マルトース、キシロース、アラビノース、フルクトース、コーンスターチやパインデックスなどのでんぷん類等の炭素源、酵母エキス、乾燥酵母、牛肉エキス、魚エキス、麦芽エキス、大豆粉、ＮＺ Ａｍｉｎｅ等の窒素源、ビオチン、チアミン、リボフラビン、ニコチン酸、ニコチン酸アミド、ピリドキシン、パントテン酸、ｐ−アミノ安息香酸等のビタミン類、ナトリウム、カリウム、亜鉛、マグネシウム、鉄（II）、鉄（III）、カルシウム、銅、リン等のミネラル類、ＦｅＳＯ4、ＭｇＳＯ4、ＣａＣＯ3、ＭｎＣｌ2、ＺｎＳＯ4、ＭｇＳＯ4、Ｋ2ＨＰＯ4、ＮＨ4ＮＯ3、ＦｅＳＯ4等の塩類を含有する培地が好ましい。培地への前記アクチノプラネス属微生物の植菌量は、最初は一白金耳のような少量でよい。培養して微生物が増殖した時点でコンパクチン又はその塩を添加するのが好ましい。 The medium used in the method of the present invention may be any medium in which Actinoplanes bacteria grow, for example, carbon sources such as starches such as glucose, maltose, xylose, arabinose, fructose, corn starch, and paraindex, yeast extract. , Dry yeast, beef extract, fish extract, malt extract, soy flour, nitrogen sources such as NZ Amine, vitamins such as biotin, thiamine, riboflavin, nicotinic acid, nicotinic acid amide, pyridoxine, pantothenic acid, p-aminobenzoic acid , Sodium, potassium, zinc, magnesium, iron (II), iron (III), minerals such as calcium, copper, phosphorus, FeSO 4 , MgSO 4 , CaCO 3 , MnCl 2 , ZnSO 4 , MgSO 4 , K 2 HPO 4, NH 4 NO 3, media containing salts of FeSO 4 and the like preferred There. The amount of the Actinoplanes spp. Inoculated into the medium may initially be as small as one platinum loop. It is preferable to add compactin or a salt thereof at the time when the microorganism grows in culture.
培地にはコンパクチン又はその塩が添加される。ここで添加とは、培地にコンパクチン又はその塩を添加してもよいし、コンパクチン若しくはその塩又はそれらを含有する溶液等に培地成分を添加してもよい。コンパクチンの塩としては、ナトリウム、カリウム、カルシウム等のアルカリ金属塩、アルカリ土類金属塩が挙げられる。培地中のコンパクチン又はその塩の濃度は、プラバスタチン又はその塩の生産性の点から、０．１ｍｇ／ｍＬ以上２．０ｍｇ／ｍＬ以下が好ましく、０．２ｍｇ／ｍＬ以上１．０ｍｇ／ｍＬ以下が好ましい。 Compactin or a salt thereof is added to the medium. Here, addition means that compactin or a salt thereof may be added to the medium, or a medium component may be added to compactin or a salt thereof, a solution containing them, or the like. Examples of the compactin salt include alkali metal salts such as sodium, potassium and calcium, and alkaline earth metal salts. The concentration of compactin or a salt thereof in the medium is preferably 0.1 mg / mL or more and 2.0 mg / mL or less, and preferably 0.2 mg / mL or more and 1.0 mg / mL or less from the viewpoint of the productivity of pravastatin or a salt thereof. preferable.
培養は、アクチノプラネス属微生物が生育する条件であればよく、例えば液体培地を用い、振とう培養、撹拌培養が好ましい。培養条件は、１５〜３５℃が好ましく、２６〜３０℃がより好ましく、ｐＨは７．５〜８．０が好ましく、７．５がより好ましい。コンパクチン又はその塩からプラバスタチン又はその塩への変換に要する時間は、３〜７日間が好ましく、３〜５日間がより好ましい。 The culture may be performed under the conditions that allow the growth of Actinoplanes microorganisms. For example, a liquid culture medium is used, and shaking culture and stirring culture are preferable. The culture condition is preferably 15 to 35 ° C, more preferably 26 to 30 ° C, and the pH is preferably 7.5 to 8.0, more preferably 7.5. The time required for converting compactin or a salt thereof to pravastatin or a salt thereof is preferably 3 to 7 days, and more preferably 3 to 5 days.
変換培養により生成したプラバスタチン又はその塩は、多くの場合精製される。精製方法としては、培養液の濃縮、沈殿化、有機溶媒による抽出、クロマトグラフィー（薄層クロマトグラフィー、カラムクロマトグラフィー、高速液体クロマトグラフィー）、および乾燥などが挙げられる。 Pravastatin or a salt thereof produced by conversion culture is often purified. Examples of the purification method include concentration, precipitation, extraction with an organic solvent, chromatography (thin layer chromatography, column chromatography, high performance liquid chromatography), and drying.
上記の方法により得られるプラバスタチンの塩としては、ナトリウム、カリウム等のアルカリ金属塩、カルシウム塩等のアルカリ土類金属塩が挙げられる。コンパクチン又はその塩からプラバスタチン又はその塩への変換率は、用いる微生物、培養条件等によって相違するが、１０％以上が好ましく、２０％以上がより好ましい。 Examples of the salt of pravastatin obtained by the above method include alkali metal salts such as sodium and potassium, and alkaline earth metal salts such as calcium salt. The conversion rate from compactin or a salt thereof to pravastatin or a salt thereof varies depending on the microorganism used, culture conditions, and the like, but is preferably 10% or more, more preferably 20% or more.
次に実施例を挙げて本発明を更に詳細に説明する。 EXAMPLES Next, an Example is given and this invention is demonstrated still in detail.
実施例１（プラバスタチン変換能の確認）
ＩＳＰ−２寒天培地（日本ベクトン・ディッキンソン株式会社製）で１４日間生育させたＲＤ０６１８０４で表されるアクチノプラネス属ＮＩＴＥ Ｐ-０１８７４株を菌体ごと掻き取り、１００ｍＬ三角フラスコ中の２０ｍＬのＢｅｎｎｅｔｔ’ｓ−マルトース培地（マルトース１０．０ｇ、酵母エキス１．０ｇ、牛肉エキス１．０ｇ、ＮＺ Ａｍｉｎｅ，Ｔｙｐｅ Ａ（和光純薬工業株式会社製）２．０ｇを蒸留水１０００ｍＬに溶解しｐＨ７．３に調整）に植菌し、２８℃、２１０ｒｐｍで４日間振盪培養を行った。
Example 1 (Confirmation of pravastatin conversion ability)
The Actinoplanes genus NITE P-01874 strain represented by RD061804, which was grown for 14 days on an ISP-2 agar medium (manufactured by Nippon Becton Dickinson Co., Ltd.), was scraped together with the bacterial cells, and 20 mL of Bennett's in a 100 mL Erlenmeyer flask. -Maltose medium (maltose 10.0 g, yeast extract 1.0 g, beef extract 1.0 g, NZ Amine, Type A (manufactured by Wako Pure Chemical Industries, Ltd.) 2.0 g was dissolved in 1000 mL of distilled water and adjusted to pH 7.3. ), And cultured with shaking at 28 ° C. and 210 rpm for 4 days.
次に、これを１００ｍＬ三角フラスコ中の２０ｍＬのＢｅｎｎｅｔｔ’ｓ−マルトース培地に５％植菌し、２８℃、２１０ｒｐｍで３日間振盪培養後、コンパクチンを終濃度０．２ｍｇ／ｍＬとなるように添加し、引き続き２８℃、２１０ｒｐｍで４日間振盪培養を行った。得られた培養液の上清をＨＰＬＣにて分析し、プラバスタチン及びコンパクチンの濃度を求めた。プラバスタチン及びコンパクチン濃度から変換率を算出した結果、プラバスタチンへの変換率は８５．３％であった。 Next, inoculate 5% of this into 20 mL of Bennett's-maltose medium in a 100 mL Erlenmeyer flask, shake culture at 28 ° C. and 210 rpm for 3 days, and then add compactin to a final concentration of 0.2 mg / mL. Subsequently, shaking culture was performed at 28 ° C. and 210 rpm for 4 days. The supernatant of the obtained culture broth was analyzed by HPLC to determine the concentrations of pravastatin and compactin. As a result of calculating the conversion rate from the concentrations of pravastatin and compactin, the conversion rate to pravastatin was 85.3%.
・ＨＰＬＣ分析条件
カラム：ＣＡＰＣＥＬＬ ＰＡＣ Ｃ１８ ＵＧ１２０ φ３ｍｍ×１５０ｍｍ
装 置：Ｗａｔｅｒｓ（株） ２６９５ Ａｌｌｉａｎｃｅ ＨＰＬＣ Ｓｙｓｔｅｍ
温 度：４０℃
移動相：Ａ液 メタノール：水：酢酸：トリエチルアミン ＝ ５００：５００：１：１
Ｂ液 １００％メタノール
Ａ液 １００％，Ｂ液 ０％ → Ａ液 ２０％，Ｂ液 ８０％となるようにグラジェント
時 間：２０分
流 速：０．７ｍＬ／ｍｉｎ
波 長：ＵＶ ２３８ｎｍ
-HPLC analysis conditions Column: CAPCELL PAC C18 UG120 φ3mm × 150mm
Equipment: Waters 2695 Alliance HPLC System
Temperature: 40 ° C
Mobile phase: Liquid A Methanol: Water: Acetic acid: Triethylamine = 500: 500: 1: 1
B liquid 100% methanol
Solution A 100%, Solution B 0% → Solution A 20%, Solution B 80% Gradient time: 20 minutes Flow rate: 0.7 mL / min
Wavelength: UV 238nm
・濃度計算方法 ・ Concentration calculation method
実施例２（プラバスタチン変換能の確認）
ＩＳＰ−２寒天培地で１４日間生育させたアクチノプラネス属に属するＲＤ０６１８０４（ＩＴＥ Ｐ-０１８７４）を菌体ごと掻き取り、１００ｍＬ三角フラスコ中の２０ｍＬの生育培地（グルコース２０．０ｇ、ピルビン酸５．０ｇ、酵母エキス３．０ｇ、大豆粉４．０ｇ、ＭｇＳＯ4・７Ｈ2Ｏ ０．２ｇ、Ｋ2ＨＰＯ4 １．５ｇ、ＮＨ4ＮＯ3 １．０ｇ、ＺｎＳＯ4・７Ｈ2Ｏ ０．０１ｇ、ＦｅＳＯ4・７Ｈ2Ｏ ０．０１ｇ、ＣａＣＯ3 ４．０ｇ、を蒸留水１０００ｍＬに溶解しｐＨ７．５に調整）に植菌し、２８℃、２１０ｒｐｍで４日間振盪培養を行った。
次に、これを１００ｍＬ三角フラスコ中の２０ｍＬの生産培地（グルコース１０．０ｇ、ピルビン酸５．０ｇ、酵母エキス３．０ｇ、大豆粉４．０ｇ、ＭｇＳＯ4・７Ｈ2Ｏ ０．２ｇ、Ｋ2ＨＰＯ4 １．５ｇ、ＮＨ4ＮＯ3 １．０ｇ、ＺｎＳＯ4・７Ｈ2Ｏ ０．０１ｇ、ＦｅＳＯ4・７Ｈ2Ｏ ０．０１ｇ、ＣａＣＯ3 ４．０ｇ、を蒸留水１０００ｍＬに溶解しｐＨ７．５に調整）に５％植菌し、２８℃、２１０ｒｐｍで３日間振盪培養後、コンパクチンを終濃度０．２ｍｇ／ｍＬとなるように添加し、引き続き２８℃、２１０ｒｐｍで４日間振盪培養を行った。実施例１と同様に変換率を求めた結果、変換率は８６．１％であった。
Example 2 (Confirmation of pravastatin conversion ability)
RD061804 (ITE P-01874) belonging to the genus Actinoplanes that was grown for 14 days on an ISP-2 agar medium was scraped together with the cells, and 20 mL of a growth medium (20.0 g of glucose, 5.0 g of pyruvic acid in a 100 mL Erlenmeyer flask. , 3.0 g of yeast extract, 4.0 g of soybean powder, 0.2 g of MgSO 4 .7H 2 O, 1.5 g of K 2 HPO 4 , 1.0 g of NH 4 NO 3 , 0.01 g of ZnSO 4 .7H 2 O, FeSO 4 · 7H 2 O 0.01g, was inoculated into CaCO 3 4.0 g, the adjustment of distilled water was dissolved in 1000mL pH7.5), 28 ℃, was carried out for 4 days shaking culture at 210 rpm.
Next, 20 mL of production medium (glucose 10.0 g, pyruvic acid 5.0 g, yeast extract 3.0 g, soybean flour 4.0 g, MgSO 4 .7H 2 O 0.2 g, K 2 in a 100 mL Erlenmeyer flask was added. Dissolve 1.5 g of HPO 4 , 1.0 g of NH 4 NO 3 , 0.01 g of ZnSO 4 .7H 2 O, 0.01 g of FeSO 4 .7H 2 O, 4.0 g of CaCO 3 in 1000 mL of distilled water, and add pH 7.5. 5% inoculated), and after shaking culture at 28 ° C. and 210 rpm for 3 days, compactin was added to a final concentration of 0.2 mg / mL, followed by shaking culture at 28 ° C. and 210 rpm for 4 days. . As a result of obtaining the conversion rate in the same manner as in Example 1, the conversion rate was 86.1%.
実施例３（コンパクチン濃度によるプラバスタチン変換能への影響）
ＩＳＰ−２寒天培地で１４日間生育させたＲＤ０６１８０４（ＮＩＴＥ Ｐ-０１８７４）を菌体ごと掻き取り、１００ｍＬ三角フラスコ中の２０ｍＬの改良生育培地（溶性でんぷん３０．０ｇ、ソヤフラワーＡ（日清オイリオグループ株式会社製）２０．０ｇ、酵母エキス１０．０ｇ、ＣａＣＯ3 ５．０ｇを蒸留水１０００ｍＬに溶解しｐＨ７．０に調整）に植菌し、２８℃、２１０ｒｐｍで４日間振盪培養を行った。
次に、これを１００ｍＬ三角フラスコ中の２０ｍＬの実施例２の生産培地に５％植菌し、２８℃、２１０ｒｐｍで３日間振盪培養後、コンパクチンを終濃度０．２、０．５及び１．０ｍｇ／ｍＬとなるように添加し、引き続き２８℃、２１０ｒｐｍで５日間振盪培養を行った。実施例１と同様に変換率を求めた結果、変換率はそれぞれ８５．４％、８２．８％及び８２．２％であった。
Example 3 (Effects of compactin concentration on pravastatin conversion ability)
RD061804 (NITE P-01874) grown for 14 days on an ISP-2 agar medium was scraped together with the cells, and 20 mL of an improved growth medium (30.0 g soluble starch, Soya Flower A (Nisshin Oillio Group) 20.0 g manufactured by the company), 10.0 g yeast extract, and 5.0 g CaCO 3 were dissolved in 1000 mL distilled water and adjusted to pH 7.0), followed by shaking culture at 28 ° C. and 210 rpm for 4 days.
Next, 5% of this was inoculated into 20 mL of the production medium of Example 2 in a 100 mL Erlenmeyer flask, and after shaking culture at 28 ° C. and 210 rpm for 3 days, the compactin concentration was 0.2, 0.5 and 1. After adding at 0 mg / mL, shaking culture was performed at 28 ° C. and 210 rpm for 5 days. As a result of obtaining the conversion rate in the same manner as in Example 1, the conversion rates were 85.4%, 82.8%, and 82.2%, respectively.
実施例４（プラバスタチン変換能に関する工業的使用の確認）
ＩＳＰ−２寒天培地で１４日間生育させたＲＤ０６１８０４（ＮＩＴＥ Ｐ-０１８７４）を菌体ごと掻き取り、１００ｍＬ三角フラスコ中の２０ｍＬの実施例３の改良生育培地に植菌し、２８℃、２１０ｒｐｍで４日間振盪培養を行った。次に、５Ｌ容ジャーファーメンター中の生産培地２．５Ｌに５％植菌し、２８℃、３５０ｒｐｍ及び１ｖｖｍ（２．５Ｌ／ｍｉｎ）で培養を行った。培養２．７日（６５時間）後にコンパクチンを終濃度１．０ｍｇ／ｍＬとなるように添加し、引き続き２８℃、３５０ｒｐｍで３日間培養を行った。実施例１と同様に変換率を求めた結果、変換率は８２．４％であった。
Example 4 (confirmation of industrial use regarding pravastatin conversion ability)
RD061804 (NITE P-01874) grown for 14 days on an ISP-2 agar medium was scraped together with the cells, inoculated into 20 mL of the improved growth medium of Example 3 in a 100 mL Erlenmeyer flask, and 4 at 28 ° C. and 210 rpm. Shaking culture was performed for 1 day. Next, 5% was inoculated into 2.5 L of production medium in a 5 L jar fermenter, and cultured at 28 ° C., 350 rpm, and 1 vvm (2.5 L / min). After culturing for 2.7 days (65 hours), compactin was added to a final concentration of 1.0 mg / mL, followed by culturing at 28 ° C. and 350 rpm for 3 days. As a result of obtaining the conversion rate in the same manner as in Example 1, the conversion rate was 82.4%.
実施例５（Ａｃｔｉｎｏｐｌａｎｅｓ同属異種菌株とのプラバスタチン変換能の比較）
独立行政法人製品評価技術基盤機構（ＮＩＴＥ）より購入したＡｃｔｉｎｏｐｌａｎｅｓ属基準株（ＮＢＲＣ株）を用いて、Ａｃｔｉｎｏｐｌａｎｅｓ属のコンパクチンをプラバスタチンに変換する能力を比較した。
ＩＳＰ−２寒天培地で１４日間生育させたＲＤ０６１８０４（ＮＩＴＥ Ｐ-０１８７４）及び各ＮＢＲＣ株を菌体ごと掻き取り、１００ｍＬ三角フラスコ中の２０ｍＬの生育培地（マルトース２０．０ｇ、ピルビン酸５．０ｇ、酵母エキス３．０ｇ、大豆粉４．０ｇ、ＭｇＳＯ4・７Ｈ2Ｏ ０．２ｇ、Ｋ2ＨＰＯ4 １．５ｇ、ＮＨ4ＮＯ3 １．０ｇ、ＺｎＳＯ4・７Ｈ2Ｏ ０．０１ｇ、ＦｅＳＯ4・７Ｈ2Ｏ ０．０１ｇ、ＣａＣＯ3 ４．０ｇ、を蒸留水１０００ｍＬに溶解しｐＨ７．５に調整）に植菌し、２８℃、２１０ｒｐｍで５日間振盪培養を行った。
次に、これを１００ｍＬ三角フラスコ中の２０ｍＬの生産培地（マルトース１０．０ｇ、ピルビン酸５．０ｇ、酵母エキス３．０ｇ、大豆粉４．０ｇ、ＭｇＳＯ4・７Ｈ2Ｏ ０．２ｇ、Ｋ2ＨＰＯ4 １．５ｇ、ＮＨ4ＮＯ3 １．０ｇ、ＺｎＳＯ4・７Ｈ2Ｏ ０．０１ｇ、ＦｅＳＯ4・７Ｈ2Ｏ ０．０１ｇ、ＣａＣＯ3 ４．０ｇ、を蒸留水１０００ｍＬに溶解しｐＨ７．５に調整）に５％植菌し、２８℃、２１０ｒｐｍで３日間振盪培養後、マルトース０．４ｇ及びコンパクチンを終濃度０．２ｍｇ／ｍＬとなるように添加し、引き続き２８℃、２１０ｒｐｍで５日間振盪培養を行った。実施例１と同様に変換率を求めた結果、表１に示したように、変換率はＲＤ０６１８０４で表されるアクチノプラネス属ＮＩＴＥ Ｐ-０１８７４株で８３．３％、ＮＢＲＣ１２５１４株で２１．４％、ＮＢＲＣ１３８７８株で３３．９％、ＮＢＲＣ１３９９９株で１０．７％であった。
Example 5 (Comparison of pravastatin conversion ability with Actinoplanes congener heterogeneous strain)
Using the Actinoplanes genus reference strain (NBRC strain) purchased from the National Institute of Technology and Evaluation (NITE), the ability to convert the Actinoplanes genus compactin into pravastatin was compared.
RD061804 (NITE P-01874) and each NBRC strain grown on an ISP-2 agar medium for 14 days were scraped together with the bacterial cells, and 20 mL growth medium (20.0 g maltose, 5.0 g pyruvic acid in a 100 mL Erlenmeyer flask, Yeast extract 3.0 g, soybean powder 4.0 g, MgSO 4 .7H 2 O 0.2 g, K 2 HPO 4 1.5 g, NH 4 NO 3 1.0 g, ZnSO 4 .7H 2 O 0.01 g, FeSO 4 -0.01 g of 7H 2 O and 4.0 g of CaCO 3 were dissolved in 1000 mL of distilled water and adjusted to pH 7.5), and shake-cultured at 28 ° C. and 210 rpm for 5 days.
Next, this was added to a 20 mL production medium (maltose 10.0 g, pyruvic acid 5.0 g, yeast extract 3.0 g, soybean flour 4.0 g, MgSO 4 .7H 2 O 0.2 g, K 2 in a 100 mL Erlenmeyer flask. Dissolve 1.5 g of HPO 4 , 1.0 g of NH 4 NO 3 , 0.01 g of ZnSO 4 .7H 2 O, 0.01 g of FeSO 4 .7H 2 O, 4.0 g of CaCO 3 in 1000 mL of distilled water, and add pH 7.5. 5%, and after shaking culture at 28 ° C. and 210 rpm for 3 days, maltose 0.4 g and compactin were added to a final concentration of 0.2 mg / mL, followed by 28 ° C. and 210 rpm for 5 days. Shaking culture was performed. As a result of obtaining the conversion rate in the same manner as in Example 1, as shown in Table 1, the conversion rate was 83.3% for Actinoplanes genus NITE P-01874 represented by RD061804, and 21.4% for NBRC12514. The NBRC 13878 strain was 33.9%, and the NBRC 13999 strain was 10.7%.
また、実施例２又は実施例３の培地を用いてＮＢＲＣ株を用いてコンパクチンからプラバスタチンへの変換能を検討した結果、Ａｃｔｉｎｏｐｌａｎｅｓ ｐｈｉｌｉｐｐｉｎｅｎｓｉｓ ＮＢＲＣ１３８７８、Ａｃｔｉｎｏｐｌａｎｅｓ ｃｏｉｃｈｉｉ ＮＢＲＣ１０６１４５、Ａｃｔｉｎｏｐｌａｎｅｓ ｒｅｇｕｌａｒｉｓ ＮＢＲＣ１２５１４、及びＡｃｔｉｎｏｐｌａｎｅｓ ｔｅｉｃｈｏｍｙｃｅｔｉｃｕｓ ＮＢＲＣ１３９９９でも、１０〜４５％の変換率が得られた。 In addition, as a result of examining the ability to convert compactin to pravastatin using the NBRC strain using the medium of Example 2 or Example 3, Actinoplanes philipinensis NBRC13878, Actinoplanes coichii NBRC106145, ActinoplanesNeclineBnecine A conversion of ˜45% was obtained.
Claims (1)
A method for producing pravastatin or a salt thereof, comprising adding compactin or a salt thereof to a medium containing RD061804 (NITE P-01874 ) belonging to the genus Actinoplanes and culturing.
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